Bottom Line:
In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS.The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants.The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants.

ABSTRACTFree radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.

fig05: Structures of DHE, EOH and ethidium and hypothesized reaction pathways to account for the formation of DHE-derived red fluorescence. EOH is basically formed from superoxide and possibly involving ONOO−/CO2 while ethidium is mainly formed from H2O2 pathways involving metal proteins. Dotted arrows indicate possible intermediate pathways involved in the formation of these products.

Mentions:
DHE is a useful fluorogenic probe for the detection of ROS including O2−•. DHE has been used increasingly as a probe for O2−• in biological systems because DHE is a hydrophobic, uncharged compound that is able to cross extra- and intracellular membranes. It undergoes significant oxidation in resting leukocytes, possibly through the uncoupling of mitochondrial oxidative phosphorylation. Cytosolic DHE displays blue fluorescence, whereas after oxidation of by oxidants such as O2−• and H2O2, it becomes 2-hydroxyethidium (2-EOH) and ethidium, which intercalates cellular DNA, staining the nucleus with a bright red fluorescence (Fig. 5). Its oxidation by different oxidizing systems has been used increasingly for fluorescent analysis of ROS output in cells and tissues. Determination of total DHE fluorescence in cells has been performed extensively in the literature for assessment of ROS and, more specifically, of O2−•. Its main drawback is that the total fluorescence of DHE is a sum of the composite spectra of all different products and thus likely reflects preferentially a measure of total cell redox state rather than production of a specific intermediate. Both 2-EOH and ethidium are fluorescent products that are difficult to discriminate them by conventional fluorescence microscopy or fluorometry. Thus, high-performance liquid chromatography (HPLC) analysis of DHE-derived fluorescent compounds (2-EOH and ethidium) has been developed in order to achieve separation and individual analysis of such products [42, 43]. This technique provides a significant increase in the accuracy of ROS output determinations and is a meaningful advance towards the precise quantification of this species in cells and tissues. Recent studies of HPLC separation and analysis of those two main products indicated that 2-EOH is generated specifically by O2−• oxidation of DHE, whereas ethidium is associated mainly with pathways involving H2O2 and metal-based oxidizing systems, including heme proteins and peroxidases [42]. More information about the DHE fluorescent probe is available in a recent review by Laurindo et al.[40]. MitoSOX™ Red mitochondrial superoxide indicator [44], a modified DHE, is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells. It is readily oxidized by superoxide inside the mitochondrion but not by other ROS- or RNS-generating systems, and oxidation of the probe is prevented by SOD. The oxidation product becomes highly fluorescent upon binding to nucleic acids [39].

fig05: Structures of DHE, EOH and ethidium and hypothesized reaction pathways to account for the formation of DHE-derived red fluorescence. EOH is basically formed from superoxide and possibly involving ONOO−/CO2 while ethidium is mainly formed from H2O2 pathways involving metal proteins. Dotted arrows indicate possible intermediate pathways involved in the formation of these products.

Mentions:
DHE is a useful fluorogenic probe for the detection of ROS including O2−•. DHE has been used increasingly as a probe for O2−• in biological systems because DHE is a hydrophobic, uncharged compound that is able to cross extra- and intracellular membranes. It undergoes significant oxidation in resting leukocytes, possibly through the uncoupling of mitochondrial oxidative phosphorylation. Cytosolic DHE displays blue fluorescence, whereas after oxidation of by oxidants such as O2−• and H2O2, it becomes 2-hydroxyethidium (2-EOH) and ethidium, which intercalates cellular DNA, staining the nucleus with a bright red fluorescence (Fig. 5). Its oxidation by different oxidizing systems has been used increasingly for fluorescent analysis of ROS output in cells and tissues. Determination of total DHE fluorescence in cells has been performed extensively in the literature for assessment of ROS and, more specifically, of O2−•. Its main drawback is that the total fluorescence of DHE is a sum of the composite spectra of all different products and thus likely reflects preferentially a measure of total cell redox state rather than production of a specific intermediate. Both 2-EOH and ethidium are fluorescent products that are difficult to discriminate them by conventional fluorescence microscopy or fluorometry. Thus, high-performance liquid chromatography (HPLC) analysis of DHE-derived fluorescent compounds (2-EOH and ethidium) has been developed in order to achieve separation and individual analysis of such products [42, 43]. This technique provides a significant increase in the accuracy of ROS output determinations and is a meaningful advance towards the precise quantification of this species in cells and tissues. Recent studies of HPLC separation and analysis of those two main products indicated that 2-EOH is generated specifically by O2−• oxidation of DHE, whereas ethidium is associated mainly with pathways involving H2O2 and metal-based oxidizing systems, including heme proteins and peroxidases [42]. More information about the DHE fluorescent probe is available in a recent review by Laurindo et al.[40]. MitoSOX™ Red mitochondrial superoxide indicator [44], a modified DHE, is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells. It is readily oxidized by superoxide inside the mitochondrion but not by other ROS- or RNS-generating systems, and oxidation of the probe is prevented by SOD. The oxidation product becomes highly fluorescent upon binding to nucleic acids [39].

Bottom Line:
In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS.The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants.The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants.

ABSTRACTFree radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.